We have previously mapped a major group specific neutralizing epitome of HIV-1 to amino acids 342-511 of gp120. We have now designed two gp120 constructs with the goal of increasing the immunogenicity without disrupting protein neutralizing or the structure of this epitome. In the first case, the conjugate is made via reductive amidation of the carbohydrate sidechains of gp120. The bond to the carrier protein is via a novel unidirectional cross-linking reagent, consisting of diamino ethane linked to bromoacetate. This reacts with the carbohydrate groups via the amino end and with free SH groups on HBsAg via the bromoacetate moiety. Conjugate formation has been demonstrated by sandwich ELISA, using antibodies specific for one antigen on the plate, and the other as the detecting reagent. Work is in progress to confirm the nature of the bond, for example, by enzymatic degradation by enzymes specific for glycoside bonds. We are also trying to determine the extent of cross-linking, by sizing chromatography by HPLC and by SDS gels. Finally, we have initiated immunogenicity testing in rabbits, looking both for the ability to induce total antibodies to gp120 by ELISA, and for the ability to induce neutralizing antibodies to the homologous nd to heterologous viral isolates in the HIV-1 plaguing assay. If successful in rabbits, we will extend these studies to rhesus monkeys and possibly to chimps, where challenge studies are possible. The second construct is the subject of a GRADA with MicroGeneSys, Inc., and the linkage is via a peptide bond. Both gp120 and HBsAg will be expressed as a large fusion protein, and the hope is that they will form particles, as HBsAg does.